Cryptococcus neoformans is an important human fungal pathogen that can cause life-threatening infections. Infections are opportunistic in nature and typically occur in immunocompromised patients, with AIDS patients being at greatest risk. Cryptococcosis is usually manifested as meningitis, which is often treated as incurable in AIDS patients and can require life-long antifungal therapy. Infections are worldwide with approximately 90- 95% of all infections being caused by serotype A strains. The importance of this fungus as a pathogen resulted in the solicitation and funding of a genome-sequencing project. The strain that was chosen, however, was a serotype D strain, which causes less than 10% of all infections. This strain was chosen because sexually compatible strains (designated MATalpha and MATa) exist, whereas compatible serotype A strains are not available due to the absence of a fertile MATa strain. An added significance of mating in C. neoformans is that virtually all infections are caused by one mating type (MATalpha). We have recently identified a serotype A MATa strain that is fertile and crosses efficiently with serotype A MATalpha strains, including strain H99, the standard laboratory strain for this serotype. The existence of this strain provides a powerful tool for studying C. neoformans because it will enable traditional genetic studies to be performed in serotype A strains. This proposal consists of three aims. The first aim will prepare two congenic pairs of strains that differ only in mating type. Genetic markers will be inserted into these strains to make them useful tools for future genetic and molecular studies. The second aim will test the role of mating type in virulence. By using congenic strains in these experiments, the role of information at the mating type locus in virulence can be tested independent of non mating-type information. In the last aim, the serotype A MATa mating type locus will be cloned and mapped to determine its size. Presently three out of the four mating type loci of C. neoformans var. neoformans (comprised of serotype A and D) are in various stages of characterization. Isolation of the serotype A MATa locus will complete this study, which is significant because of the role that mating type plays in virulence.